The development of immunogens capable of eliciting broadly neutralizing antibodies to HIV-1 remains one of the most difficult challenges confronting biomedical research today. Nonetheless, a small number of human monoclonal antibodies (mAbs) have been isolated from HIV-1-infected individuals that can neutralize primary isolates of HIV-1 from different genetic subtypes. Of these rare mAbs, two (2F5 and 4E10) recognize epitopes on the membrane-proximal region of the gp41 ectodomain. However, all attempts to elicit comparable anti-gp41 antibodies by vaccination with peptides or simple linear epitopes have thus far failed. Our approach to immunogen design is guided by our recent identification and structural determination of a novel two-stranded coiled-coil domain (C55) that includes the membrane-proximal epitope region of gp41. However, we have determined that the C55 dimer structure is too unstable to be suitable for immunogenicity studies. The overall goal of this research plan is to gain a detailed understanding of the structural and thermodynamic properties of the C55 coiled-coil domain, and to use this knowledge to design and produce stable immunogens for the elicitation of broadly neutralizing HIV-1 antibodies. Our central hypothesis is that presentation of the highly conserved sequences recognized by 2F5 and 4E10 in this new structural state opens a promising avenue for the discovery and development of novel immunogens to induce broadly reactive neutralizing antibodies. Specific aims of this research are: (1) To identify and develop stabilized versions of the C55 coiled-coil domain encompassing the 2F5 and 4E10 epitopes. We will identify and incorporate apolar side chain substitutions into the buried hydrophobic core of the dimer to stabilize the coiled-coil structure. We will also screen cysteine substitutions to introduce a disulfide bond between the two interacting helices. Our emphasis is to generate stable molecules that preserve both the dimer conformation and the critical surface-exposed residues of the membrane-proximal region. (2) To evaluate the immunological responses elicited by stabilized C55 coiled-coil variants in small animals. We will conduct immunogenicity studies in rabbits and guinea-pigs to determine whether the stabilized dimer structure can elicit neutralizing antibodies. These studies will involve immunization of the animals using soluble proteins. We will also evaluate the immunogenicity of the stable coiled-coil molecules captured onto nanometer-sized beads as particulate immunogens. [unreadable] [unreadable] [unreadable]